molecules Article Thymol Mitigates Cadmium Stress by Regulating Glutathione Levels and Reactive Oxygen Species Homeostasis in Tobacco Seedlings Xiefeng Ye 1,†, Tianxiao Ling 1,†, Yanfeng Xue 2,†, Cunfa Xu 3, Wei Zhou 4, Liangbin Hu 4, Jian Chen 5,6,* and Zhiqi Shi 5,6,* 1 College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China; [email protected] (X.Y.); [email protected] (T.L.) 2 Nanjing Yangzi Modern Agriculture Investment and Development Co. Ltd., Nanjing 211899, China; [email protected] 3 Central Laboratory, Jiangsu Academy of Agricultural Science, Nanjing 210014, China; [email protected] 4 Department of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China; [email protected] (W.Z.); [email protected] (L.H.) 5 Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China 6 Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Jiangsu Provincial Department of Agriculture and Forestry, Nanjing 210014, China * Correspondence: [email protected] (J.C.); [email protected] (Z.S.); Tel.: +86-25-8439-1863 (J.C.) † These authors contribute equally to this work. Academic Editor: Derek J. McPhee Received: 10 August 2016; Accepted: 1 October 2016; Published: 14 October 2016 Abstract: Thymol is a famous plant-derived compound that has been widely used in pharmacy due to its antioxidant and antimicrobial properties. However, the modulation of intrinsic plant physiology by thymol remains unclear. It is a significant challenge to confer plant tolerance to Cd (cadmium) stress. In the present study physiological, histochemical, and biochemical methods were applied to investigate thymol-induced Cd tolerance in tobacco (Nicotiana tabacum) seedlings. Thymol was able to alleviate Cd-induced growth inhibition of tobacco seedlings in both dose- and time-dependent manners. Both histochemical detection and in-tube assays suggested that thymol treatment blocked Cd-induced over-generation of reactive oxygen species (ROS), lipid peroxidation, and loss of membrane integrity in both leaves and roots. Thymol decreased Cd-induced cell death that was indicated in vivo by propidium iodide (PI) and trypan blue, respectively. Thymol stimulated glutathione (GSH) biosynthesis by upregulating the expression of γ-glutamylcysteine synthetase 1 (GSH1) in Cd-treated seedlings, which may contribute to the alleviation of Cd-induced oxidative injury. In situ fluorescent detection of intracellular Cd2+ revealed that thymol significantly decreased free Cd2+ in roots, which could be explained by the thymol-stimulated GSH biosynthesis and upregulation of the expression of phyochelatin synthase 1 (PCS1). Taken together, these results suggested that thymol has great potential to trigger plant resistant responses to combat heavy metal toxicity, which may help our understanding of the mechanism for thymol-modulated cell metabolic pathways in response to environmental stimuli. Keywords: thymol; reactive oxygen species; cell death; glutathione; cadmium; tobacco 1. Introduction Large amounts of Cd (cadmium) have been released into the environment by both natural and anthropogenic process, which has been drawing great attention worldwide [1]. In the Cd-contaminated agricultural environment, the ionic cadmium (Cd2+) can be readily taken up by plants, leading to Molecules 2016, 21, 1339; doi:10.3390/molecules21101339 www.mdpi.com/journal/molecules Molecules 2016, 21, 1339 2 of 15 phytotoxicity and posing potential risk to human health through the food chain [2]. One of the most important toxic effects of Cd stress on plants is to induce the accumulation of reactive oxygen species (ROS), which further triggers oxidative injury, stress-responsive signaling, and cell death [3]. Among the resistant strategies developed by plants, glutathione (GSH) plays dual functions in combating Cd toxicity. First, GSH acts as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging Cd-induced ROS in plants [4]. Second, GSH is an indispensable precursor for the biosynthesis of phytochelatins (PCs). PCs are composed of small cysteine-rich peptides, which can chelate and compartmentalize Cd to attenuate cytotoxicity induced by free Cd in plant cells [5,6]. Both the GSH-biosynthetic gene GSH1 (g-glutamylcysteine synthetase 1) and the PCs-biosynthetic gene PCS1 (phyochelatin synthase 1) have been demonstrated to be critical for Cd detoxification in plants [7,8]. Remediation of Cd-contaminated soils is the fundamental method to solve the problem of Cd-induced eco-toxicity, but it is hard to apply extensively due to some disadvantages (such as high cost or being time-consuming) [9]. Recently, exogenous regulation of intrinsic plant physiology has been recommended as a simple alternative to decrease Cd-induced phytotoxicity in Cd-contaminated environment [10,11]. Thymol [5-methyl-2-(1-methylethyl) phenol] is the main constituent of oils obtained from Thymus vulgaris. Thymol is considered as a natural monoterpene phenol that has been highly appreciated for its clinical relevance and antimicrobial activity [12,13]. It has been reported that thymol shows anti-inflammatory and anti-oxidative properties by triggering immune responses in mammalian cells [14–16]. Thymol can be applied as a food preservative to maintain the quality of fruits and vegetables during postharvest storage [17,18]. Based on the evaluation of the U.S. Environmental Protection Agency (EPA) Office of Pesticide Programs, thymol has minimal potential toxicity and poses minimal risk [19]. In addition, the U.S. Food and Drug Administration (FDA) has listed thymol as a Generally Recognized as Safe (GRAS) substance [20]. Therefore, thymol has great potential to be applied as a safe bio-agent in agriculture. Although thymol is able to protect mammalian cells from environmental stimuli [21], whether and how thymol can modulate intrinsic plant physiology is still elusive. In this study, we investigated the protecting effect of thymol on Cd-induced growth inhibition in tobacco (Nicotiana tabacum) seedlings. The role of thymol in the attenuation of ROS accumulation and oxidative injury in Cd-treated seedlings was further confirmed. Finally, the role of thymol-induced endogenous GSH biosynthesis was studied. The possible mechanism of thymol driving these physiological processes, and their significance, are discussed as well. 2. Results 2.1. Thymol Significantly Alleviated Cd-Induced Growth Inhibition of Tobacco Seedlings Treatment with CdCl2 remarkably inhibited the root growth of tobacco seedlings in a dose-dependent manner. The roots were exposed to CdCl2 at 0–80 µM for 72 h. Compared to the control, the root length decreased by 22.05%, 30.04%, 42.21%, 52.85%, and 64.14% at 5, 10, 20, 40, and 80 µM of Cd levels, respectively (Figure1A). Treatment with Cd at 20 µM was used for further experiments. To understand the effect of thymol on the regulation of root growth under Cd stress, thymol with different concentrations (0–400 µM) were added to the treatment solution. Under normal conditions without Cd stress, thymol treatment inhibited the root growth of seedlings. However, compared to Cd treatment alone, the addition of thymol at 50–200 µM resulted in the significant increase in root length. Thymol at 100 µM showed the greatest effect on the alleviation of Cd-induced inhibition of root elongation (Figure1B). In a time-course experiment, the Cd-induced decrease in root length was significantly recovered when roots were incubated in the treatment solution containing both Cd and 100 µM of thymol. At the end of the experiment (up to 72 h), the root length under Cd + thymol treatment significantly increased by 35.10% as compared to Cd treatment alone (Figure1C). In addition, thymol significantly enhanced the fresh weight of both shoots and roots under Cd stress (Figure1D). Thymol treatment alone showed a slight inhibitory effect on seedling growth (Figure1B–D), but these results suggested that thymol recovered the growth of tobacco seedlings from Cd stress. Molecules 2016, 21, 1339 3 of 15 Molecules 2016, 21, 1339 3 of 14 FigureFigure 1. 1.The The effect effect of of thymol thymol on on the thegrowth growth ofof tobaccotobacco seedlings under under Cd Cd stress. stress. (A ()A The) The roots roots of of seedlings were treated with CdCl2 at 0–80 μM for 72 h for the measurement of root length; (B) In the seedlings were treated with CdCl2 at 0–80 µM for 72 h for the measurement of root length; (B) In the presence of CdCl2 at 20 μM, the roots of seedlings were treated with thymol at different concentrations presence of CdCl2 at 20 µM, the roots of seedlings were treated with thymol at different concentrations (0–400 μM) for 72 h. Then the root length was measured; (C) The roots of seedlings were exposed to (0–400 µM) for 72 h. Then the root length was measured; (C) The roots of seedlings were exposed 20 μM of CdCl2 and 100 μM of thymol simultaneously for 6, 12, 24, 48, and 72 h, respectively, for the to 20 µM of CdCl2 and 100 µM of thymol simultaneously for 6, 12, 24, 48, and 72 h, respectively, for measurement of root length; (D) The roots of seedlings were treated with water, 20 μM of CdCl2, 100 μM the measurement of root length; (D) The roots of seedlings were treated with water, 20 µM of CdCl , of thymol, alone, or their combinations for 72 h. Then the shoots and roots were harvested, respectively, 2 100 µM of thymol, alone, or their combinations for 72 h. Then the shoots and roots were harvested, for the quantification of fresh weight. Each value was represented as the mean of three replicates with SD. respectively, for the quantification of fresh weight. Each value was represented as the mean of three Different letters in (B) and (D) indicate that the mean values are significantly different between the replicatestreatments with (p < SD.0.05, Different ANOVA, lettersLSD). Different in (B) and letters (D in) indicate(C) indicate that that the the mean mean valuesvalues are are significantly significantly differentdifferent between among thefour treatments treatments (p at< a 0.05, particular ANOVA, point LSD).